The present invention relates to the use of particulate contrast agents in various diagnostic imaging techniques based on light, more particularly to particulate light imaging contrast agents.
Contrast agents are employed to effect image enhancement in a variety of fields of diagnostic imaging, the most important of these being X-ray, magnetic resonance imaging (MRI), ultrasound imaging and nuclear medicine. Other medical imaging modalities in development or in clinical use today include magnetic source imaging and applied potential tomography. The history of development of X-ray contrast agents is almost 100 years old.
The X-ray contrast agents in clinical use today include various water-soluble iodinated aromatic compounds comprising three or six iodine atoms per molecule. The compounds can be charged (in the form of a physiologically acceptable salt) or non-ionic. The most popular agents today are non-ionic substances because extensive studies have proven that non-ionic agents are much safer than ionics. This has to do with the osmotic loading of the patient. In addition to water-soluble iodinated agents, barium sulphate is still frequently used for X-ray examination of the gastrointestinal system. Several water-insoluble or particulate agents have been suggested as parenteral X-ray contrast agents, mainly for liver or lymphatic system imaging. Typical particulate X-ray contrast agents for parenteral administration include for example suspensions of solid iodinated particles, suspensions of liposomes containing water-soluble iodinated agents or emulsions of iodinated oils.
The current MRI contrast agents generally comprise paramagnetic substances or substances containing particles (hereinafter xe2x80x9cmagnetic particlesxe2x80x9d) exhibiting ferromagnetic, ferrimagnetic or superparamagnetic behaviour. Paramagnetic MRI contrast agents can for example be transition metal chelates and lanthanide chelates like Mn EDTA and Gd DTPA. Today, several gadolinium based agents are in clinical use; including for example Gd DTPA (Magnevist(copyright)), Gd DTPA-BMA (Omniscan(copyright)), Gd DOTA (Dotarem(copyright)) and Gd HPDO3A (Prohance(copyright)). Several particulate paramagnetic agents have been suggested for liver MRI diagnosis; for example suspensions of liposomes containing paramagnetic chelates and suspensions of paramagnetic solid particles like for example gadolinium starch microspheres. Magnetic particles proposed for use as MR contrast agents are water-insoluble substances such as Fe3O4 or xcex4-Fe2O3 optionally provided with a coating or carrier matrix. Such substances are very active MR contrast agents and are administered in the form of a physiologically acceptable suspension.
Contrast agents for ultrasound contrast media generally comprise suspensions of free or encapsulated gas bubbles. The gas can be any acceptable gas for example air, nitrogen or a perfluorocarbon. Typical encapsulation materials are carbohydrate matrices (e.g. Echovist(copyright) and Levovist(copyright)), proteins (e.g. Albunex(copyright)), lipid matrials like phospholipids (gas-containing liposomes) and synthetic polymers.
Markers for diagnostic nuclear medicine like scintigraphy generally comprise radioactive elements like for example technetium (99m) and indium (III), presented in the form of a chelate complex, whilst lymphoscintigraphy is carried out with radiolabelled technetium sulphur colloids and technetium oxide colloids.
The term xe2x80x9clight imagingxe2x80x9d used here includes a wide area of applications, all of which utilize an illumination source in the UV, visible or IR regions of the electromagnetic spectrum. In light imaging, the light, which is transmitted through, scattered by or reflected (or re-emitted in the case of fluorescence) from the body, is detected and an image is directly or indirectly generated. Light may interact with matter to change its direction of propagation without significantly altering its energy. This process is called elastic scattering. Elastic scattering of light by soft tissues is associated with microscopic variations in the tissue dielectric constant. The probability that light of a given wavelength (xcex) will be scattered per unit length of travel in tissue is termed the (linear) scattering coefficient xcexcs. The scattering coefficient of soft tissue in an optical window of approx. 600-1300 nm ranges from 101-103 cmxe2x88x921 and decreases as 1/xcex. In this range xcexcs greater than  greater than xcexca (the absorption coefficient) and although xcexcs (and the total attenuation) is very large, forward scattering gives rise to substantial penetration of light into tissue. Ballistic light is light that has travelled through a region of tissue without being scattered. Quasi-ballistic light (xe2x80x9csnakexe2x80x9d light) is scattered light that has maintained approximately the same direction of travel. The effective penetration depth shows a slow increase or is essentially constant with increasing wavelengths above 630 nm (although a slight dip is observed at the water absorption peak at 975 nm). The scattering coefficient shows only a gradual decrease with increasing wavelength.
Light that is scattered can either be randomly dispersed (isotropic) or can scatter in a particular direction with minimum dispersion (anisotropic) away from the site of scattering. For convenience and mathematical modelling purposes, scattering in tissue is assumed to occur at discrete, independent scattering centers (xe2x80x9cparticlesxe2x80x9d). In scattering from such xe2x80x9cparticlesxe2x80x9d, the scattering coefficient and the mean cosine of scatter (phase function) depend on the difference in refractive index between the particle and its surrounding medium and on the ratio of particle size to wavelength. Scattering of light by particles that are smaller than the wavelength of the incident light is called Rayleigh scattering. This scattering varies as 1/xcex4 and the scattering is roughly isotropic. Scattering of light by particles comparable to or larger than the wavelength of light is referred to as Mie scattering. This scattering varies as 1/xcex and the scattering is anisotropic (forward peaked). In the visible/near-IR where most measurements have been made, the observed scattering in tissue is consistent with Mie-like scattering by particles of micron scale: e.g. cells and major organelles.
Since the scattering coefficient is so large for light wavelengths in the optical window (600-1300 nm), the average distance travelled by a photon before a scattering event occurs is only 10-100 xcexcm. This suggests that photons that penetrate any significant distance into tissue encounter multiple scattering events. The ballistic component of light that has travelled several centimeters through tissue is exceedingly small. Multiple scattering in tissue means that the true optical path length is much greater than the physical distance between the light input and output sites. The scattering acts, therefore, to diffuse light in tissue (diffuse-transmission and -reflection). The difficulty that multiple scattering presents to imaging is three-fold: (i) light that has been randomized due to multiple scattering has lost signal information and contributes noise to the image (scattering increases noise); (ii) scattering keeps light within tissue for a greater period of time, increasing the probability for absorption, so less light transmits through tissue for detection (scattering decreases signal); and (iii) the determination of physical properties of tissue (or contrast media) such as concentration that could be obtained from the Beer-Lambert law is complicated since the true optical path length due to scattering is difficult to determine (scattering complicates the quantification of light interactions in tissue). However, although light cannot penetrate more than a few tens of microns in tissue without being scattered, the large value of the mean cosine of scattering indicates that a significant fraction of photons in an incident beam may undergo a large number of scatters without being deviated far from the original optical axis, and as such can contribute in creating an image. As a result, it can be possible to perform imaging on tissue despite the predominance of scatter, if the noise component can be rejected and the quasi-ballistic component of the light can be detected.
The most interesting wavelengths for light imaging techniques are in the approximate range of 600-1300 nm. These wavelengths have the ability to penetrate relatively deeply into living tissue without absorption by natural substances and furthermore are harmless to the human body. However, for optical analysis of surface structures or diagnosis of diseases very close to the body surface or body cavity surfaces or lumens, UV light and visible light below 600 nm wavelength can also be used.
Light can also be used in therapy; thus for example in Photodynamic Therapy (PDT) photons are absorbed and the energy is transformed into heat and/or photochemical reactions which can be used in cancer therapy.
The main methods of light imaging today include simple transillumination, various tomographic techniques, fluorescence imaging, and hybrid methods that involve irradiation with or detection of other forms of radiation or energy in conjunction with irradiation with or detection of light (such as photoacoustic or acousto-optical). These methods take advantage of either transmitted, scattered or emitted (fluorescence) photons or a combination of these effects. The present invention relates to contrast agents for any of these and further imaging methods based on any form of light.
There is today great interest in development of new equipment for imaging based on light. Interesting methods are especially the various types of tomographic techniques in development especially in Japan. As scientific references to the use of light in diagnostic medicine and PDT see for example Henderson, B. and Dougherty, T. in Photodynamic Therapy. 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There are several patent publications which relate to light imaging technology and to the use of various dyes in light imaging: a labeling fluorescent dye comprising hydroxy aluminium 2,3-pyrido cyanide in JP 4,320,456 (Hitachi Chem), therapeutic and diagnostic agent for tumors containing fluorescent labelled phthalocyanine pigment in JP 4288 022 (Hitachi Chem), detection of cancer tissue using visible native luminescence in U.S. Pat. No. 4,930,516 (Alfano R. et al.), method and apparatus for detection of cancer tissue using native fluorescence in U.S. Pat. No. 5,131,398 (Alfano, R. et al.), improvements in diagnosis by means of fluorescenct light emmision from tissue in WO 90/10219 (Andersson-Engels, S. et al.), fluorescent porphyrin and fluorescent phthalocyanine-polyethylene glycol, polyol, and saccharide derivatives as fluorescent probes in WO91/18006 (Diatron Corp), method of imaging a random medium in U.S. Pat. No. 5,137,355 (State Univ. of New York), tetrapyrrole therapeutic agents in U.S. Pat. No. 5,066,274 (Nippon Petrochemicals), tetrapyrrole polyaminomonocarboxylic acid in therapeutic agents in U.S. Pat. No. 4,977,177 (Nippon Petrochemicals), tetrapyrrole aminocarboxylic acids in U.S. Pat. No. 5,004,811 (Nippon Petrochemicals), porphyrins and cancer treatment in U.S. Pat. No. 5,162,519 (Efamol Holdings), dihydroporphyrins and method of treating tumors susceptible to necrosis in U.S. Pat. No. 4,837,221 (Efamol), parenterally administered zinc phthalocyanide compounds in form of liposome dispersion containing synthetic phospholipids in EP 451 103 (CIBA Geigy), apparatus and method for detecting tumors in U.S. Pat. No. 4,515,165 (Energy Conversion Devices), time and frequency domain spectroscopy determining hypoxia in WO92/13598 (Nim Inc), phthalocyanatopolyethylene glycol and phthalocyanato saccharides as fluorescent digoxin reagent in WO 91/18007 (Diatron), fluorometer in U.S. Pat. No. 4,877,965 (Diatron), fiberoptic fluorescence spectrometer in WO 90/00035 (Yale Univ.), tissue oxygen measuring system in EP 502,270 (Hamamatsu Photonics), method for determining bilirubin concentration from skin reflectance in U.S. Pat. No. 4,029,084 (Purdue Research Foundation), bacteriochlorophyll-a derivative useful in photodynamic therapy in U.S. Pat. No. 5,173,504 (Health Research Inc), purified hematoporphyrin dimers and trimers useful in photodynamic therapy in U.S. Pat. No. 5,190,966 (Health Research Inc), drugs comprising porphyrins in U.S. Pat. No. 5,028,621 (Health Research Inc), hemoporphyrin derivatives and process of preparing in U.S. Pat. No. 4,866,168 (Health Research Inc), method to destroy or impair target cells in U.S. Pat. No. 5,145,863 (Health Research Inc), method to diagnose the presence or absence of tumor tissue in U.S. Pat. No. 5,015,463 (Health Research Inc), photodynamic therapeutic technique in U.S. Pat. No. 4,957,481 (U.S. Bioscience), apparatus for examining living tissue in U.S. Pat. No. 2,437,916 (Philip Morris and Company), transillumination method apparatus for the diagnosis of breast tumors and other breast lesions by normalization of an electronic image of the breast in U.S. Pat. No. 5,079,698 (Advanced Light Imaging Technologies), tricarbocyanine infrared absorbing dyes in U.S. Pat. No. 2,895,955 (Eastman Kodak), optical imaging system for neurosurgery in CA 2,048,697 (Univ. Techn. Int.), new porphyrin derivatives and their metallic complexes as photosensitizer for PDT in diagnosis and/or treatment of cancer in JP 323,597 (Hogyo,T), light receiving system of heterodyne detection and image forming device for light transmission image in EP 445,293 (Research Development Corp. of Japan), light receiving system of heterodyne detection and image forming device for light transmission image using light receiving system in WO 91/05239 (Research Development Corp. of Japan), storage-stable porphyrin compositions and a method for their manufacture in U.S. Pat. No. 4,882,234 (Healux), method for optically measuring chemical analytes in WO 92/19957 (Univ. of Maryland at Baltimore), wavelength-specific cytotoxic agents in U.S. Pat. No. 4,883,790 (Univ. of British Columbia), hydro-monobenzo-porphyrin wavelength-specific cytotoxic agents in U.S. Pat. No. 4,920,143 (Univ. of British Columbia), apparatus and method for quantitative examination and high-resolution imaging of human tissue in EP 447,708 (Haidien Longxing Med Co), optical imaging system for neurosurgery in U.S. Pat. No. 7,565,454 (University Technologies Int. Inc.), xe2x80x94characterization of specific drug receptors with fluorescent ligands in WO 93/03382 (pharmaceutical Discovery Corp), 4,7-dichlorofluorescein dyes as molecular probes in U.S. Pat. No. 5,188,934 (Applied Biosystems), high resolution breast imaging device utilizing non-ionizing radiation of narrow spectral bandwith in U.S. Pat. No. 4,649,275 (Nelson, R. et al.), meso-tetraphenyl-porphyrin-Komplexverbindungen, Verfaren zu ihrer Herstellung und Diese Enthaltends Pharmazeutische Mittel in EP 336,879 (Schering), 13,17-propionsaure und propionsaurederivat Substituerte Porphyrin-Komplexverbindungen, Verfahren zu ihrer Herstellung und diese Enthaltende Pharmazeutische Mittel in EP 355,041 (Schering), photosensitizing agents in U.S. Pat. No. 5,093,349 (Health Research), pyropheophorbides and their use in photodynamic therapy in U.S. Pat. No. 5,198,460 (Health Research), optical histochemical analysis, in vivo detection and real-time guidance for ablation of abnormal tissues using Raman spectroscopic detection system in WO 93/03672 (Redd, D.), tetrabenztriazaporphyrin reagents and kits containing the same in U.S. Pat. No. 5,135,717 (British Technology Group), system and method for localization of functional activity in the human brain in U.S. Pat. No. 5,198,977 (Salb, J.). photodynamic activity of sapphyrins in U.S. Pat. No. 5,120,411 (Board of Regents, University of Texas), process for preparation of expanded porphyrins in U.S. Pat. No. 5,152,509 (Board of Regents, University of Texas), expanded porphyrins (Board of Regents, University of Texas), infrared radiation imaging system and method in WO 88/01485 (Singer Imaging), imaging using scattered and diffused radiation in WO 91/07655 (Singer Imaging), diagnostic apparatus for intrinsic fluorescence of malignant tumor in U.S. Pat. No. 4,957,114, indacene compounds and methods for using the same in U.S. Pat. No. 5,189,029 (Bo-Dekk Ventures), method of using 5,10,15,20-tetrakis (carboxy phenyl) porphine for detecting cancers of the lung in U.S. Pat. No. 5,162,231 (Cole, D. A. et al.), Verfahren zur Abbildung eines Gewebebereiches in DE 4327 798 (Siemens), chlorophyll and bacteriochlorophyll derivatives, their preparation and pharmaceutical compositions comprising them in EPO 584 552 (Yeda Research and Development Company), wavelength-specific photosensitive porphacyanine and expanded porphyrin-like compounds and methods for preparation and use thereof in WO 94/10172 (Qudra Logic Technologies), method and apparatus for improving the signal to noise ratio of an image formed of an object hidden in or behind a semiopaque random media in U.S. Pat. No. 5,140,463 (Yoo, K. M. et al.), benzoporphyrin derivatives for photodynamic therapy in U.S. Pat. No. 5,214,036 (University of British Columbia), fluorescence diagnostics of cancer using delta-amino levulinic acid in WO 93/13403 (Svanberg et al.), Verfahren zum Diagnostizieren von mit fluoreszierenden Substansen angereicherten, inbesondere tumorxc3x6sen Gewebebereichen in DE 4136 769 (Humboldt Universitxc3xa4t), terpyridine derivatives in WO 90/00550 (Wallac).
All the light imaging dyes or contrast agents described in the state-of-the-art have different properties, but all those agents have an effect on the incident light, leading to either absorption and/or fluorescence. However none of these contrast agents is used as a particulate contrast agent.
We have now found that contrast enhancement may be achieved particularly efficiently in light imaging methods by introducing particulate materials as scattering contrast agents. For the sake of clarity, the word xe2x80x9cparticlexe2x80x9d is used to refer to any physiologically acceptable particulate materials. Such particles may be solid (e.g. coated or uncoated crystalline materials) or fluid (e.g. liquid particles in an emulsion) or may be aggregates (e.g. fluid containing liposomes). Particulate material with a particle size smaller than or similar to the incident light wavelength are preferred.
Thus viewed from one aspect the invention provides the use of a physiologically tolerable particulate material for the manufacture of a particulate-contrast-agent containing contrast medium for use in in vivo dignostic light imaging.
Viewed from a further aspect the invention also provides a method of generating an image of the human or non-human (preferably mammalian, avian or reptilian) animal body by light imaging, characterised in that a contrast effective amount of a physiologically tolerable particulate contrast agent is administered to said body, and an image of at least part of said body is generated. In such a method a contrast effective amount of the particulate agent is administered, e.g. parenterally or into an externally voiding body organ or duct, light emitted, transmitted or scattered by the body is detected and an image is generated of at least part of the body in which the contrast agent is present. Hybrid methods in which light, either alone or in conjunction with other forms of radiation, is administered to the body, and light, or some other form of radiation, is detected. In particular, the other form of radiation may be ultrasound.
The particles used according to the invention are preferably water-insoluble or at least sufficiently poorly soluble as to retain their desired particle size (e.g. 600-1300 nm) for at least 2 hours following administration into the body under investigation.
The images generated may be spatial or temporal and mono- or multi-dimensional.
In a further aspect of the invention, the imaging technique may be used to determine a value for a parameter characteristic of the body or the part of the body under study, e.g. blood flow rate. In this case however, the parameter determination should be based on light detected from particles studied through the skin or through an endoscopically or surgically exposed surface.
Particularly preferably, the light imaging procedure used is selected from confocal scanning laser microscopy (CSLM), optical coherence tomography (OCT), laser doppler, laser speckle, and multi-photon microscopy techniques (for a description of the latter see for example Denk, W. in Photonics Spectra (1997) July 125-130, Denk, W. et al. in Science (1990) April 248 73-76, Denk, W. et al. in J.Neurosci.Meth. (1994) 54:2:151-162, Denk, W. et al. in Neuron (1997) January 18:351-357, Maiti, S. et al. in Science (1997) January 275 530-532 and Denk, W. et al. in Proc. Natl. Acad. (1995) August 92:18:8279-8282).
Confocal scanning laser microscopy (CSLM) is an imaging modality that selectively detects a single point within a test object by focusing light from a pinhole source onto that point. The light transmitting past or reflecting from that point is refocused onto a second pinhole that filters out light coming from any other site in the object except the focal point. Raster scanning of the focus point through a plane passing through the sample generates a full image of that plane of points. Moving the pinholes and focusing apparatus back and forth from the sample selects out different sample planes. In effect CSLM is a means for xe2x80x9copticallyxe2x80x9d sectioning a test sample. It pulls out images of individual sections of the sample, but without the necessity that those sections be physically separated from the rest of the sample.
Optical coherence tomography (OCT) accomplishes optical sectioning in a related, but somewhat different manner. A collimated beam of light is reflected from the sample, then is compared with a reference beam that has travelled a precisely known distance. Only the light travelling exactly the same distance to the sample and back as the distance the reference beam travels from the source to the detector constructively interferes with the reference beam and is detected. Thus the light from a single plane within the sample is again selected. Varying the distance that the reference beam travels before it is compared with the sampling beam selects out different sample planes.
CSLM, OCT, laser doppler and laser speckle are discussed for example by: Rajadhyaksha et al. in Laser Focus World, February 1997, pages 119 to 127; Sabel et al. in Nature Medicine 3(2): 244-247 (1997); Tearney et al. in SPIE 2389: 29-34 (1995); Bonner et al. in xe2x80x9cScattering techniques applied to supramolecular and non-equilibrium systemsxe2x80x9d, pages 685-701, Ed. Chen et al., Plenum; Ruth in J. Microcirc: Clin Exp 9: 21-45 (1990); Pierard in Dermatology 186: 4-5 (1993); and Bonner et al. in xe2x80x9cLaser-doppler blood flowmetryxe2x80x9d pages 17 to 46, Ed. Shepherd et al., Kluwer, 1990.
CSLM and OCT may be used particularly effectively to study structures and events occurring in the skin or within about a millimeter of an accessible surface of the body under study, e.g. a surface exposed during surgical operation or exposed endoscopically.
CSLM and OCT can be useful in optically guided tumor resection. For example, either device attached to a colonoscope may facilitate determination that no residual malignant tissue remains after removal of a cancerous colon polyp. Additional applications include, but are not limited to, diagnosis and treatment of disease in the rest of the digestive tract, surgical treatment of ulcerative colitis, and diagnosis and treatment of endometriosis.
Dynamically, CSLM and OCT can be used to follow the movement of blood cells through the capillaries of the skin and other vascularized tissue lying within about a millimeter of an exposed surface. Potentially they can also be used in conjunction with laser Doppler or speckle inferferometry for the measure of blood flow.
Laser Doppler and speckle interferometry are related, each relying upon the fact that the intensity of light detected after a beam of laser light that interacts with a collection of moving particles changes with time. Mathematical analysis of the changes provides a basis for calculating the rate at which the particles are moving.
The perfusion of tissue that is exposed by surgery is one important indicator of the health of that tissue. Blood flow within the skin of the breast may be an indicator of internal disease. Blood flow in the skin can be detected by laser Doppler blood-flow measurement or laser speckle interferometry, either by itself or in conjunction with CSLM or OCT.
According to the present invention, synthetic particles, capable of scattering light of the wavelength used for the imaging procedure, may be administered as contrast agents in an in vivo light imaging procedure. Typically such scattering particles will be administered in suspension in a physiologically tolerable fluid (e.g. water for injections, physiological saline, Ringer""s solution etc.) into the vasculature or musculature or into the tissue or organ of interest.
A preferred contrast agent for intraoperative CSLM or OCT will have the following properties: it will consist of stabilized particles in an aqueous or buffered liquid medium. The particle size will preferably be around 600 to 1300 nm, more preferably 700 to 1100 nm (i.e. roughly equal to the wavelength of the light source). The refractive index of the particles will preferably differ from that of body fluids, such as blood and lymph, by at least 0.01. Optionally the particles may have fluorescent dyes attached to their surfaces or contained within them or the particles themselves may be composed of fluorescent dyes. Optionally the particles may have suitable surface modifying agents, such as poly(ethylene glycol), to slow their uptake by macrophages in the body and to prolong their blood circulation lifetimes.
The particles may be of a material which is transparent or translucent or more preferably opaque to light of the wavelength of the light source.
Particularly preferably, the particles are substantially monodisperse polymer particles (with a coefficient of variation of the particle size (i.e. 100xc3x97 standard deviation÷mean particle size by volume of the major mode of the detectable particles) as measured by a Coulter LS 130 particle size analyzer of less than 10%, preferably less than 5%). Such particles may be prepared by the SINTEF technique disclosed in U.S. Pat. No. 4,336,173 and U.S. Pat. No. 4,459,378. Such polymer particles may be simple scatterers or may be modified to carry a chromophore (or fluorophore), preferably having characteristic absorption and/or emission maxima in the 600 to 1300 nm range. Furthermore they may be modified to include or carry a targetting vector, e.g. a species serving to cause the particles to accumulate at a desired target site, for example superparamagnetic crystals which allow the particle to be accumulated at a target site by application of an external magnetic field, or a drug, antibody, antibody fragment or peptide (e.g. an oligopeptide or polypeptide) which has a binding affinity for sites within the target zone, e.g. cell surface receptors.
The particulate contrast agent can be applied through simple topical application or other pharmaceutically acceptable routes. For dermatological applications, the contrast agents may be modified to be delivered through transdermal patches or by iontophoretesis. Iontophoretic delivery is preferred, as one can control the amount of the agent that is delivered.
For intraoperative uses the contrast agent can be injected into the vasculature or into the lesion to be removed prior to or during the surgery. For detection of lymph nodes it can be injected into a lymph duct draining into the surgical area. Alternatively it may be applied during surgery as a topical ointment, a liquid, or a spray. For measurement of blood flow the agent can be injected intravascularly prior to the measurement.
As indicated above, the particulate agents used according to the invention may comprise a chromophore or fluorophore, i.e. may absorb or emit light in the wavelength range detected in the imaging procedure or alternatively may rely primarily upon light scattering effects. In the latter case, one may simply use physiologically tolerable non photo-labelled particles, e.g. particles of an inert organic or inorganic material, e.g. an insoluble triiodophenyl compound or titanium dioxide, which appears white or colourless to the eye. Where the particles comprise a fluorophore or chromophore, i.e. are photo-labelled, this may be in a material carried by (e.g. bound to, coated on, or contained or deposited within) a particulate carrier (e.g. a solid particulate or a liposome). Alternatively the carrier itself may have chromophoric or fluorophoric properties. While the photolabel may be a black photolabel (i.e. one which absorbs across the visible spectrum and thus appears black to the eye) non-black photolabels are preferred.
Scattering contrast agents (and absorbing contrast agents for that matter) can have several mechanisms in image enhancement for light imaging applications. The first mechanism is a direct image enhancing role similar to the effect that x-ray contrast media have in x-ray imaging. In direct image enhancement, the contrast medium contributes directly to an improvement in image contrast by affecting the signal intensity emanating from the tissue containing the contrast medium. In light imaging, scattering (and absorbing) agents localized in a tissue can attenuate light differently than the surrounding tissue, leading to contrast enhancement.
For near surface methods such as confocal microscopy and optical coherence tomography, scattering agents generate contrast primarily by serving as reflection centres that selectively direct the incident light to the detector. When scattering sites are trapped in a moving fluid, such as blood, the extent of the scattering sites"" movement can be used as a measure of the fluid""s flowrate.
The xe2x80x9cspecklexe2x80x9d phenomenon results from the interaction of coherent radiation (such as that from a laser) with scattering sites. When the scattering sites move, the speckle pattern changes with time, and the rate of change of the speckle pattern can be used to determine the rate of movement of the scattering sites. If the movement of the scattering sites is non-random, for example when they are entrained in a moving fluid, the rate of fluid flow can be determined by the changes in the speckle pattern over time.
A second mechanism by which a scattering (or absorbing) agent could be used is as a noise rejection agent. The contrast agent in this case is not directly imaged as described above, but functions to displace a noise signal from an imaging signal so that the desired signal is more readily detected. Noise in light imaging applications results from multiple scattering and results in a degradation of image quality. The origin of this noise is as follows:
As previously mentioned, light propagating through a random medium such as tissue undergoes multiple scattering. This scattering splits the incident light into three components, the ballistic, quasi-ballistic, and incoherent (highly scattered) components. The ballistic and quasi-ballistic signals propagate through tissue in the forward direction and carry the object information. The incoherent component constitutes noise because the light has undergone random scattering in all directions and information about the object is lost. When the intensity of the ballistic and quasi-ballistic signals are reduced below the intensity of the multiply scattered noise, the object becomes invisible. This multiple scattering noise can be partially removed by a spatial filter that rejects light scattered away from the collinear direction of the incident light. However, a substantial portion of noise emerges from the object after multiple scattering events by rejoining the original ballistic signal. This multiply scattered light can not be removed by spatial filtering due to its collinear path with the desired ballistic signal.
Scattering (and absorbing) agents can aid in the removal of unwanted noise component from the desired ballistic and quasi-ballistic signals. This is based on the fact that multiply scattered light undergoes a random walk in tissue and thus travels over a longer path length than the ballistic signal. The distance the ballistic and quasi-ballistic signals traverses is essentially the thickness of the tissue (or body part) being imaged. Scattered light traveling a longer distance has a greater probability of being attenuated. Current technology uses a time-gate (temporal filter) to reject the scattered signal (longer traveling= longer residence time in tissue) from the ballistic and quasi-ballistic components.
The introduction of a small isotropic scattering agent greatly increases the residence time of the highly scattered signal component while having a lesser effect on the ballistic and quasi-ballistic components. This effectively provides a longer separation between the ballistic and quasi-ballistic signals and the highly scattered component, providing improved rejection of the scattered (noise) component and better image quality.
Very little is disclosed in prior art regarding particulate scattering-based contrast agents. To our knowledge the only prior art with regard to particulate scattering-based contrast agents is U.S. Pat. No. 5,140,463 (Yoo, K. M. et al.) which discloses a method and apparatus for improving the signal to noise ratio of an image formed of an object hidden in or behind a semi-opaque medium. The patent in general terms suggests to make the random medium less random (so that there will be less scattered light) and it is also suggested to increase the time separation between ballistic and quasi-ballistic light and the highly scattered light. One of many ways to obtain this will, according to the patent, be to introduce small scatterers into the random medium. There are no further suggestions regarding these small scatterers and no suggestion of in vivo use.
Particulate materials in the form of liposomes have been suggested; liposome or LDL-administered Zn(II)-phthalocyanine has been suggested as photodynamic agent for tumors by Reddi, E. et al. in Lasers in Medical Science 5 (1990) 339, parenterally administered zinc phtalocyanine compositions in form of liposome dispersion containing synthetic phopholipid in EP 451 103 (CIBA Geigy) and liposome compositions containing benzoporphyrin derivatives used in photodynamic cancer therapy or an antiviral agents in CA 2,047,969 (Liposome Company). These particulate materials have been suggested as therapeutic agents and have nothing to do with scattering light imaging contrast agents.
In one embodiment of the invention the contrast medium for imaging modalities based on light will comprise physiologically tolerable gas containing particles. Preferred are e.g. biodegradable gas-containing polymer particles, gas-containing liposomes or aerogel particles.
This embodiment of the invention includes, for example, the use in light imaging of particles with gas filled voids (U.S. Pat. No. 4,442,843), galactose particles with gas (U.S. Pat. No. 4,681,119), microparticles for generation of microbubbles (U.S. Pat. No. 4,657,756 and DE 3313947), protein microbubbles (EP 224934), clay particles containing gas (U.S. Pat. No. 5,179,955), solid surfactant microparticles and gas bubbles (DE 3313946), gas-containing microparticles of amylose or polymer (EP 327490), gas-containing polymer particles (EP 458079), aerogel particles (U.S. Pat. No. 5,086,085), biodegradable polyaldehyde microparticles (EP 441468), gas associated with liposomes (WO 9115244), gas-containing liposomes (WO 9222247), and other gas containing particles (WO 9317718, EP 0398935, EP 0458745, WO 9218164, EP 0554213, WO 9503835, DE 3834705, WO 9313809, WO 9112823, EP 586875, WO 9406477, DE 4219723, EP 554213, WO 9313808, WO 9313802, DE 4219724, WO 9217212, WO 9217213, WO 9300930, U.S. Pat. No. 5,196,183, WO 9300933, WO 9409703, WO 9409829, EP 535387, WO 9302712, WO 9401140). The surface or coating of the particle can be any physiologically acceptable material and the gas can be any acceptable gas or gas mixture. Specially preferred gases are the gases used in ultrasound contrast agents like for example air, nitrogen, lower alkanes and lower fluoro or perfluoro alkanes (e.g. containing up to 7, especially 4, 5 or 6 carbons).
Where gas microbubbles (with or without a liposomal encapsulating membrane) are used according to the invention, advantage may be taken of the known ability of relatively high intensity bursts of ultrasound to destroy such microbubbles. Thus by comparing the detected light signal (or image) before and after ultrasound exposure mapping the distribution of the contrast agent may be facilitated.
In another embodiment of the invention the contrast medium for imaging modalities based on light will comprise physiologically tolerable particles of lipid materials, e.g. emulsions, especially aqueous emulsions. Preferred are halogen comprising lipid materials. This embodiment of the invention includes, for example, the use in light imaging of fat emulsions (JP 5186372), emulsions of fluorocarbons (JP 2196730, JP 59067229, JP 90035727, JP 92042370, WO 930798, WO 910010, EP 415263, WO 8910118, U.S. Pat. No. 5,077,036, EP 307087, DE 4127442, U.S. Pat. No. 5,114,703), emulsions of brominated perfluorocarbons (JP 60166626, JP 92061854, JP 5904630, JP 93001245, EP 231070), perfluorochloro emulsions (WO 9311868) or other emulsions (EP 321429).
In yet another embodiment of the invention the contrast medium for imaging modalities based on light will comprise physiologically tolerable liposomes. Preferred groups of liposomes are phospholipid liposomes and multilamelar liposomes.
This embodiment of the invention includes, for example, the use in light imaging of phospholipid liposomes containing cholesterol derivatives (U.S. Pat. No. 4,544,545); liposomes associated with compounds containing aldehydes (U.S. Pat. No. 4,590,060); lipid matrix carriers (U.S. Pat. No. 4,610,868); liposomes containing triiodobenzoic acid derivatives of the type also suitable for X-ray examination of liver and spleen (DE-2935195); X-ray contrast liposomes of the type also suitable for lymphography (U.S. Pat. No. 4,192,859); receptor-targeted liposomes (WO-8707150); immunoactive liposomes (EP-307175); liposomes containing antibody specific for antitumor antibody (U.S. Pat. No. 4,865,835); liposomes containing oxidants able to restore MRI contrast agents (spin labels) which have been reduced (U.S. Pat. No. 4,863,717); liposomes containing macromolecular bound paramagnetic ions of the type also suitable for MRI (GB-2193095); phospholipid liposomes of the type also suitable for ultrasound imaging containing sodium bicarbonate or aminomalonate as gas precursor (U.S. Pat. No. 4,900,540); stable plurilamellar vesicles (U.S. Pat. No. 4,522,803); oil-filled paucilamellar liposomes containing non-ionic surfactant as lipid (U.S. Pat. No. 4,911,928); liposomal phospholipid polymers containing ligands for reversible binding with oxygen (U.S. Pat. No. 4,675,310); large unilamellar vesicle liposomes containing non-ionic surfactant (U.S. Pat. No. 4,853,228); aerosol formulations containing liposomes (U.S. Pat. No. 4,938,947 and U.S. Pat. No. 5,017,359); liposomes containing amphipathic compounds (EP-361894); liposomes produced by adding an aqueous phase to an organic lipid solution followed by evaporating the solvent and then adding aqueous lipid phase to the concentrate (FR-2561101); stable monophasic lipid vesicles of the type also useful for encapsulation of bioactive agents at high concentrations (WO-8500751); homogeneous liposome preparations (U.S. Pat. No. 4,873,035); stabilized liposome compounds comprising suspensions in liquefiable gel (U.S. Pat. No. 5,008,109); lipospheres (solid hydrophilic cores coated with phospholipid) of the type also suitable for controlled extended release of active compounds (WO-9107171); liposomes sequestered in gel (U.S. Pat. No. 4,708,861); metal chelates bound to liposomes, also suitable for use as MR contrast agents (WO-9114178); lipid complexes of X-ray contrast agents (WO-8911272); liposomes which can capture high solute to lipid ratios (WO-9110422); liposomes containing covalently bound PEG moieties on external surface to improve serum half-life (WO-9004384); contrast agents comprising liposomes of specified diameter encapsulating paramagnetic and/or superparamagnetic agents (WO-9004943); liposomes of the type also suitable for delivering imaging agents to tumours consisting of small liposomes prepared from pure phopholipids (EP-179444); encapsulated X-ray contrast agents such as iopromide in liposomes (U.S. Pat. No. 5,110,475); non-phospholipid liposome compositions (U.S. Pat. No. 5,043,165 and U.S. Pat. No. 5,049,389); hepatocyte-directed vesicle delivery systems (U.S. Pat. No. 4,603,044); gas-filled liposomes of the type also suitable as ultrasound contrast agents for imaging organs (U.S. Pat. No. 5,088,499); injectable microbubble suspensions stabilized by liposomes (WO-9115244); paramagnetic chelates bound to liposomes (U.S. Pat. No. 5,135,737); liposome compositions of the type also suitable for localising compounds in solid tumors (WO-9105546); injectable X-ray opacifying liposome compositions (WO-8809165); encapsulated iron chelates in liposomes (EP-494616); liposomes linked to targeting molecules through disulphide bonds (WO-9007924); and compositions consisting of non-radioactive crystalline X-ray contrast agents and polymeric surface modifiers with reduced particle size (EP-498482). Water soluble compounds which, in simple aqueous solution are not apparently significant light scatterers or absorbers, may become efficient scatterers on incorporation within liposomes. Thus iodixanol (and other soluble iodinated X-ray contrast agents that are commercially available) provides a clear solution on dissolution in water. However when iodixanol is encapsulated in liposomes the resulting particulate product is off-white indicating a significant light scattering capability.
Besides using liposomes as carriers for light imaging contrast agents, it is possible to use simple micelles, formed for example from surfactant molecules, such as sodium dodecyl sulphate, cetyltrimethylammonium halides, pluronics, tetronics etc., as carriers for photolabels which are moderately or substantially water insoluble but are solubilised by the amphiphilic micelle forming agent, e.g. photolabels such as indocyanine green. Similarly peptides such as PEG modified polyaspartic acid (see Kwon et al. Pharm. Res. 10: 970 (1993)) which spontaneously aggregate into polymeric micelles may be used to carry such photolabels. Likewise photolabel carrier aggregate particles can be produced by treatment of polycyclic aromatic hydrocarbons with anionic surfactants (e.g. sodium dodecyl sulphate or sulphated pluronic F108) and subsequent addition of heavy metal ions (e.g. thorium or silver). Such heavy metal treatment gives rise to micelles exhibiting phosphorescent behaviour and these can be used in the present invention without incorporation of a photolabel, especially using a pulsed light source and gated detection of the temporally delayed phosphorescent light.
In a still further embodiment of the invention the contrast medium for imaging modalities based on light will comprise physiologically tolerable particles containing iodine. These particles may for example be particles of a substantially water insoluble solid or liquid iodine-containing compound, e.g. an inorganic or organic compound, in the latter case preferably a triiodophenyl group containing compound, or alternatively they may be aggregate particles (such as liposomes) in which at least one of the components is an iodinated compound. In this case the iodinated compound may be a membrane forming compound or may be encapsulated by the membrane. For example, the use of emulsified iodinated oils (U.S. Pat. No. 4,404,182), particulate X-ray contrast agents (JP 67025412, SU 227529, DE 1283439, U.S. Pat. No. 3,368,944, AU 9210145, EP 498482, DE 4111939, U.S. Pat. No. 5,318,767), iodinated esters (WO 9007491, EP 300828, EP 543454, BE 8161143) and iodinated lipids (EP 294534) are included in this embodiment of the invention.
In a yet still further embodiment of the invention the contrast medium for imaging modalities based on light will comprise physiologically tolerable magnetic particles. The term xe2x80x9cmagnetic particlexe2x80x9d as used here means any particle displaying ferromagnetic, ferrimagnetic or superparamagnetic properties and preferred are composite particles comprising magnetic particles and a physiologically tolerable polymer matrix or coating material, e.g. a carbohydrate and/or a blood residue prolonging polymer such as a polyalkyleneoxide (e.g. PEG) as described for example by Pilgrimm or Illum in U.S. Pat. No. 5,160,725 and U.S. Pat. No. 4,904,479 e.g. biodegradable matrix/polymer particles containing magnetic materials.
This embodiment of the invention includes, for example, the use in light imaging of magnetic liquid (SU 1187221), ferrite particles coated with a negatively charged colloid (DE 2065532), ferrite particles (U.S. Pat. No. 3832457), liquid microspheres containing magnetically responsive substance (EP 42249), magnetic particles with metal oxide core coated with silane (EP 125995), magnetic particles based on protein matrix (DE 3444939), magnetic vesicles (JP 60255728), magnetic particles (SU 106121), magnetic particles embedded in inert carrier (JP 62167730), ferromagnetic particles loaded with specific antibodies (DE 3744518), superparamagnetic particles coated with biologically acceptable carbohydrate polymers (WO 8903675), polymerized lipid vesicles containing magnetic material (U.S. Pat. No. 4,652,257), superparamagnetic materials in biodegradable matrices (U.S. Pat. No. 4,849,210), biodegradable matrix particles containing paramagnetic or ferromagnetic materials (U.S. Pat. No. 4,675,173), ferromagnetic particles with substances for binding affinity for tissue (WO 8601112), ferrite particles (JP 47016625, JP 47016624), ferromagnetic particles (NL 6805260), magnetic polymer particles (WO 7800005, JP 62204501, JP 94016444, WO 870263), barium ferrite particles (WO 8805337), magnetic iron oxide particles (U.S. Pat. No. 4,452,773), amino acid polymer containing magnetic particles (U.S. Pat. No. 4,247,406), complexed double metal oxide particles (EP 186616), magnetic particles (GB 2237198), encapsulated superparamagnetic particles (WO 8911154), biodegradable magnetic particles (WO 8911873), magnetic particles covalently bond to proteins (EP 332022), magnetic particles with carbohydrate matrix (WO 8301768), magnetic particles with silicon matrix (EP 321322), polymer coated magnetic particles (WO 9015666), polymer-protected collodial metal dispersion (EP 252254), biodegradable superparamagnetic particles (WO 8800060), coated magnetic particles (WO 9102811), ferrofluid (DE 4130268), organometallic coated magnetic particles (WO 9326019) and other magnetic particles (EP 125995, EP 284549, U.S. Pat. No. 5,160,726, EP 516252, WO 9212735, WO 9105807, WO 9112025, WO 922586, U.S. Pat. No. 5,262,176, WO 9001295, WO 8504330, WO 9403501, WO 9101147, EP 409351, WO 9001899, EP 600529, WO 9404197).
The particulate contrast agent used according to the invention may, as mentioned above, be non-photo-labelled or photolabelled. In the latter case this means that the particle either is an effective photoabsorber at the wavelength of the incident light (i.e. carries a chromophore) or is a fluorescent material absorbing light of the incident wavelength and emitting light at a different wavelength (i.e. carries a fluorophore). Examples of suitable fluorophores include fluorescein and fluorescein derivatives and analogues, indocyanine green, rhodamine, triphenylmethines, polymethines, cyanines, phalocyanines, naphthocyanines, merocyanines, lanthanide complexes (e.g. as in U.S. Pat. No. 4,859,777) or cryptates, etc. including in particular fluorophores having an emission maximum at a wavelength above 600 nm (e.g. fluorophores as described in WO-A-92/08722). Other labels include fullerenes, oxatellurazoles (e.g. as described in U.S. Pat. No. 4,599,410), LaJolla blue, porphyrins and porphyrin analogues (e.g. verdins, purpurins, rhodins, perphycenes, texaphyrins, sapphyrins, rubyrins, benzoporphyrins, photofrin, metalloporphyrins, etc.) and natural chromophores/fluorophores such as chlorophyll, carotenoids, flavonoids, bilins, phytochrome, phycobilins, phycoerythrin, phycocyanins, retinoic acid and analogues such as retinoins and retinates.
In general, photolabels which contain chromophores should exhibit a large molar absorptivity, e.g.  greater than 105 cmxe2x88x921Mxe2x88x921 and an absorption maximum in the optical window 600 to 1300 nm. Particulates for use as noise rejection agents by virtue of their absorption properties should similarly preferably have molar absorptivities in excess of 105 cmxe2x88x921Mxe2x88x921 and an absorption maximum in the range 600 to 1300 nmxe2x88x921. For fluorescent particles, the quantum yield for fluorescence is one of the most important characteristics. This should be as high as possible. However the molar absorptivity should also desirably be above 105 cmxe2x88x921Mxe2x88x921 for the fluorophore and the absorption maximum should desirably be in the range 600 to 1300 nm for diffuse reflectance studies or 400 to 1300 nm for surface studies.
These photo-labelled materials may be used as such if substantially water-insoluble and physiologically tolerable, e.g. as solid or liquid particles, or alternatively may be conjugated to or entrapped within a particulate carrier (e.g. an inorganic or organic particle or a liposome). Particularly preferred in this are conjugates of formula I
I3Phxe2x88x92Lxe2x88x92C*xe2x80x83xe2x80x83(I) 
where I3Ph is a triiodophenyl moiety, L is a linker moiety and C* is a chromophore or fluorophore (e.g. as described above). Such compounds form a further aspect of the invention.
The I3Ph moiety is preferably a 2, 4, 6 triiodo moiety having carboxyl or amine moieties (or substituted such moieties, e.g. alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, alkoxycarbonylalkoxycarbonyl, or alkylcarbonylamino groups where the alkyl or alkylene moieties are optionally hydroxy substituted and preferably contain up to 20, particularly 1 to 6, especially 1 to 3 carbons) at the 3 and 5 positions. The linker group L may be any group capable of linking the group C* to the I3Ph moiety, e.g. an amide, amine, NHSO2 or carboxyl group or a thio analog thereof; or a C1-2Oalkylene chain terminated by such groups and optionally with one or more methylene groups replaced by thia or oxa and optionally substituted for example by thio, oxo, hydroxy or alkyl moieties. Examples of group L include xe2x80x94NHSO2xe2x80x94 and xe2x80x94CO2(CH2)2Oxe2x80x94CSxe2x80x94NHxe2x80x94.
Such compounds may be prepared by conjugating a chromophoric or fluorophoric molecule to a triiodophenyl compound of the type proposed as X-ray contrast agents by Nycomed, Sterling Winthrop, or Bracco in their numerous patent publications (by way of example U.S. Pat. No. 5,264,610, U.S. Pat. No. 5,328,404, U.S. Pat. No. 5,318,767 and U.S. Pat. No. 5,145,684).
In one particular embodiment of the invention, non-photolabelled particles, e.g. solid particles of a polymer or an iodinated X-ray contrast agent, are provided with a coating or shell of a photolabel, e.g. a fluorescent agent, for example by chemically or physiochemically binding the photolabel to the particles (e.g. by using oppositely charged photolabel and particles). The resulting coated particles, preferably of nano particle size (e.g. 5 to 800 nm, especially 10 to 500 nm) if labelled with a fluorophore would allow light energy trapped by the core to be transferred to the luminescing surface and so enhance light emission by the fluorophore. Compositions containing such particles form a further aspect of the invention.
Alternatively the photo-label may be entrapped within a solid polymer matrix, e.g. by co-precipitation of polymer and photolabel or by precipitation of photo-label within the pores of a porous inorganic or organic matrix.
Suitable organic polymer matrices for use as carriers or cores for photolabels are substantially water insoluble physiologically tolerable polymers, e.g. polystyrene latex, polylactide coglycolide, polyhydroxybutyrate co-valerate etc.
Other physiologically acceptable particles may be used in contrast media for imaging methods based on light in accordance with of the present invention. Preferred groups of materials are e.g. biodegradable polymer particles, polymer or copolymer particles and particles containing paramagnetic materials. The particles can for example be crosslinked gelatin particles (JP 60222046), particles coated with hydrophilic substances (JP 48019720), brominated perfluorocarbon emulsions (JP 58110522), perfluorocarbon emulsions (JP 63060943), particles and emulsions for oral use (DE 3246386), polymer particles (WO 8601524, DE 3448010), lipid vesicles (EP 28917), metal oxide particles (JP 1274768), metal transferrin dextran particles (U.S. Pat. No. 4,735,796), monodisperse magnetic polymer particles (WO 8303920), polymer particles (DE 2751867), microparticles containing paramagnetic metal compounds (U.S. Pat. No. 4,615,879), porous particles containing paramagnetic materials (WO 8911874), hydrophilic polymer particles (CA 1109792), water-swellable polymer particles (DE 2510221), polymer particles (WO 8502772), metal loaded molecular sieves (WO 9308846), barium sulphate particles (SU 227529), metal particles (DE 2142442), crosslinked polysaccharide particles (NL 7506757), biodegradable polymer particles (BE 869107), niobium particles (SU 574205), biodegradable polymer particles (EP 245820), amphiphilic block copolymers (EP 166596), uniform size particles (PT 80494), coloured particles (WO 9108776), polymer particles (U.S. Pat. No. 5,041,310, WO 9403269, WO 9318070, EP 520888, DE 4232755), porous polymer particles (WO 9104732), polysaccharide particles (EP 184899), lipid emulsions (SU 1641280), carbohydrate particles (WO 8400294), polycyanoacrylate particles (EP 64967), paramagnetic particles (EP 275215), polymer nanoparticles (EP 240424), nanoparticles (EP 27596, EP 499299), nanocapsules (EP 274961), inorganic particles (EP 500023, U.S. Pat. No. 5,147,631, WO 9116079), polymer particles ((EP 514790), apatite particles (WO 9307905), particulate micro-clusters (EP 546 939), gel particles (WO 9310440), hydrophilic colloids (DE 2515426), particulate polyelectrolyte complex (EP 454044), copolymer particles (EP 552802), paramagnetic polymer particles (WO 9222201), hydrophilic poly-glutamate microcapsules (WO 9402106) and other particles (WO 9402122, U.S. Pat. No. 4,997,454, WO 9407417, EP 28552, WO 8603676, WO 8807870, DE 373809, U.S. Pat. No. 5,107,842, EP 502814).
In general, where the particulate agent is intended for parenteral administration (e.g. into the vasculature), it may be desirable to prolong the blood residence time for the particles by attaching to these a blood residence time prolonging polymer as described for example by Pilgrimm in U.S. Pat. No. 5,160,725 or Illum in U.S. Pat. No. 4,904,479. In this way imaging of the vascular system may be facilitated by delaying the uptake of the particle by the reticuloendothelial system. In the case of liposomal particles, the blood residence prolonging polymer may be bound to preformed liposomes or, conjugated to liposomal membrane forming molecules, may be used as an amphiphilic membrane forming component so resulting in liposomes carrying the hydrophilic blood residence polymer component on their surfaces. Alternatively or additionally the particles may be conjugated to a biotargetting moiety (e.g. as described in WO-A-94/21240) so as to cause the particles to distribute preferentially to a desired tissue or organ, e.g. to tumor tissue.
The particle size utilized according to the invention will depend upon whether particle administration is parenteral or into an externally voiding body cavity and on whether or not the particles are photo-labelled. In general particle sizes will be in the range 5 to 10000 nm, especially 15 to 1500 nm, particularly 50 to 400 nm and for particles which are being used for their scattering effect particle size will preferably be in the range {fraction (1/15)} to 2xcex, or more preferably {fraction (1/10)}xcex to xcex, especially xcex/4Π to xcex/Π, more especially about xcex/2Π (where xcex is the wavelength of the incident light in the imaging technique). By selecting a particle size which scatters effectively at wavelengths above the absorption maxima for blood, e.g. in the range 600 to 1000 nm, and by illuminating at a wavelength in that range, the contrast efficacy of non-photolabelled particles may be enhanced.
For administration to human or animal subjects, the particles may conveniently be formulated together with conventional pharmaceutical or veterinary carriers or excipients. The contrast media used according to the invention may conveniently contain pharmaceutical or veterinary formulation aids, for example stabilizers, antioxidants, osmolality adjusting agents, buffers, pH adjusting agents, colorants, flavours, viscosity adjusting agents and the like. They may be in forms suitable for parenteral or enteral administration, for example, injection or infusion or administration directly into a body cavity having an external voidance duct, for example the gastrointestinal tract, the bladder and the uterus. Thus the media of the invention may be in conventional pharmaceutical administration forms such as tablets, coated tablets, capsules, powders, solutions, suspensions, dispersions, syrups, suppositories, emulsions, liposomes, etc; solutions, suspensions and dispersions in physiologically acceptable carrier media, e.g. water for injections, will however generally be preferred. Where the medium is formulated for parenteral administration, the carrier medium incorporating the particles is preferably isotonic or somewhat hypertonic.
The contrast agents can be used for light imaging in vivo, in particular of organs or ducts having external voidance (e.g. GI tract, uterus, bladder, etc.), of the vasculature, of phagocytosing organs (e.g. liver, spleen, lymph nodes, etc.) or of tumors. The imaging technique may involve endoscopic procedures, e.g. inserting light emitter and detector into the abdominal cavity, the GI tract etc. and detecting transmitted, scattered or reflected light, e.g. from an organ or duct surface. Where appropriate monochromatic incident light may be utilized with detection being of temporally delayed light emission (e.g. using pulsed light gated detection) or of light of wavelengths different from that of the incident light (e.g. at the emission maximum of a fluorophore in the contrast agent). Similarly images may be temporal images of a selected target demonstrating build up or passage of contrast agent at the target site. The light used may be monochromatic or polychromatic and continuous or pulsed; however monochromatic light will generally be preferred, e.g. laser light. The light may be ultraviolet to near infra-red, e.g. 100 to 1300 nm wavelength however wavelengths above 300 nm and especially 600 to 1000 nm are preferred.
The contrast media of the invention should generally have a particle concentration of 1xc2x710xe2x88x926 g/ml to 50xc2x710xe2x88x923 g/ml, preferably 5xc2x710xe2x88x926 g/ml to 10xc2x710xe2x88x923 g/ml. Dosages of from 1xc2x710xe2x88x927 g/kg to 5xc2x710xe2x88x921 g/kg, preferably 1xc2x710xe2x88x926 g/kg to 5xc2x710xe2x88x922 g/kg will generally be sufficient to provide adequate contrast although dosages of 1xc2x710xe2x88x924 g/kg to 1xc2x710xe2x88x922 g/kg will normally be preferred.
The various publications referred to herein are hereby incorporated by reference.
The invention is further illustrated by the following non-limiting Examples. Unless otherwise stated percentages and ratios are by weight.